The molecular mechanism of clathrin-mediated endocytosis is highly conserved among eukaryotes. The WASP/WIP/myosin module regulates actin assembly to generate forces that invaginate the plasma membrane and pinch off endocytic vesicles. A multidisciplinary approach combining live-cell imaging, yeast genetics and actin biochemistry will be used to investigate this process. Ultimate goals are reconstitution of actin assembly-driven membrane invagination and vesicle scission events on artificial liposomes, and activation and ultrastructural analysis of purified contractile rings. These studies will elucidate functions of individual cytoskeletal proteins and will provide a systems level understanding of the interplay among the proteins of each complex system. The ability to dissect the individual steps of actin-mediated endocytosis in live cells by image analysis, combined with reconstitution of WASP-mediated endocytic actin assembly on microbead surfaces in cell extracts, will make attainable a comprehensive understanding of how actin assembly is initiated, harnessed for vesicle formation, and terminated. Actin assembly in extracts will be reconstituted on membrane surfaces to investigate both the roles of type 1 myosins in driving membrane invagination, and the roles of actin assembly and BAR proteins in driving vesicle scission. Theoretical modeling will generate hypotheses for future experiments. Candidates for the protein factors that regulate and mediate disassembly will be added to our extract system to reconstitute biological disassembly activity. How the same actin subunit assembles into distinct filament-based structures with vastly different organization and associated protein composition will be investigated in the extract system by EM and protein mass spectrometry. Particular attention will be focused upon the myosins, since a different isoform associates with each of the three actin structures despite sharing a common, conserved ATPase domain. The first successful isolation of the contractile apparatus from a genetically tractable organism will be exploited for EM ultrastructural analysis of isolated wild-type and mutant structures, and a full investigation of the contractile mechanism, its regulation, and the ring disassembly pathway will be investigated.